Study On H₂O₂ And Ethylene Mediating The Effect Of Nitrogen Forms Induced Rhizosphere PH Change To Rice Iron Nutrion And Growth

Rice(Oryza sativa)is one of the most important crops in the world,which is widely planted and accounts for approximately 1/3 of the cereal crops planting area.NH4+ and NO3-are the two major nitrogen(N)sources that are taken up by plant roots.Rice has a strong ammonium assimilation ability and ammonium is better for rice growth than nitrate,so it is generally recognized that rice is a typical preferring ammonium species.But the reason is little known.In this research,from the perspective of differential physiological,transcriptional and metabolic responses to high pH,our results demonstrated that nitrate uptake-induced rhizosphere alkalization confers rice preference for ammonium.And we verified that H2O2 and ethylene regulated these physiological,transcriptional and metabolic changes,all which work together to influence rice sensitivity to alkaline stress.The main results are followed below:1 The adaption of rice to ammonium is related to its acidic performanceRice is ammonium preference crop,as sole nitrate nutrient severely inhibited rice growth,which manifest new leaf chlorosis and iron plaque formation on root surface.When controlled the pH 5.5 by adjusting the solution pH everyday with nitrate,the phenotype of leaf chlorosis and iron plaque did not occur and the rice grown well as the plants in ammonium nutrient.Compared with upland crops that use nitrate as the main source of nitrogen,we found that rice did not grow under alkaline conditions,but showed better adaption to acid conditions.In contrast,the upland crops grow well in the alkaline conditions.Together,these results suggest that the rice preference of ammonium and not nitrate is tightly related to the external pH in the growth environment.The adaption of plants to ammonium or nitrate may be related to its growth conditions where is acidic or alkaline.2 High pH induced physiological and molecular iron deficiency responseIron deficiency is responsible for the leaf chlorosis under high pH condition,as foliar spraying of Fe could recover the leaves from chlorosis.Under the high pH condition,the expression of metal transporter genes including OsNRAMPl,OsIRT1 and OsIRT2,and OsYSL15 and OsYSL2 was markedly increased.The expressions of OsNAS1,OsNAS2,OsNAS3 and OsNAATl in the roots were 1.1,2.7,1.8 and 2.0 times higher than those in plants at a pH 6.The genes encoding NAS and NAAT are critical in the biosynthesis of MA from L-Met.A significant induction of OsIR02,which preferentially binds to the upstream sequences of Fe deficiency inducible genes,such as OsNAS1,OsNAS3,OsIRTl and OsYSL15 was also observed for roots in the pH 8 culture solution.The strong up-regulation of these genes responding to the Fe deficiency suggests the enhanced capacity for the uptake and transport of Fe in rice roots under a high pH condition and provided the evidences for plant Fe deficiency induced by a high pH.3 H2O2 enhanced the root oxidizability under high pH conditionIn this study,the occurrence of Fe deficiency in rice is tightly related to iron plaque formation on the root surface under the high pH condition,where Fe availability is strongly decreased.Aerenchyma develops upon cell death and lysis,which is mediated by reactive oxygen species(ROS),especially H2O2.H2O2 induced radial oxygen loss(ROL),which then oxidizes the rhizosphere Fe2+ into Fe3+,e.g.,4Fe2++ O2+ 6H2O=4Fe(OH)3+ 8H+.Meanwhile,H2O2 induced the expression of Prxs and POD activity,which may directly participate in iron plaque formation through a Haber-Weiss-type reaction:Fe2+ + H2O2 = Fe3+ + OH-+ OH-.4 H2O2 regulated the phenylpropanoid which hegatively affected the apoplast Fe mobilizationTotal phenolic compounds in root tissue are quickly increased in response to both external low pH and high pH,but the iron binding capacity is much higher in phenolic compounds extracted from pH 4 conditions.Further results showed that increasing the external pH leads to a dramatic reduction in the cell wall fluorescent intensity of phenolic compounds and the secretion of phenolic substances.The LC-MS data verified that the phenols of cinnamic acid,caffeic acid and protocatechuic acid were strongly decreased by a high pH of 7.5.All of these results suggested that the external pH affected the phenolic compounds.From the digital gene expression data,we found the phenylpropanoid metabolism was the strongly disturbed pathway.Genes(PAL,C4H,4CL,CCR,CAD,Prx)encoding enzymes that mediate the biosynthesis of phenylpropanoids entering lignin synthesis(via conversion into coniferaldehyde)were down-regulated at pH 4.However,these genes were induced at pH 8,suggesting that root phenolic compounds changed through the phenylpropanoid pathway.The histological staining then demonstrated that lignin formation was enhanced by a high external pH.All of the above suggested that the phenolic compounds were changed under the high pH condition through the up-regulation of genes involved in phenylpropanoid metabolism.5 Ethylene negatively affected rice alkali-resistance by inhibited the PM H+-ATPasePM H+-ATPase is important for the root proton-secretion adaptation to alkaline stress.However,our observed that alkaline stress inhibited the H+-ATPase activity evaluated by the H+ efflux rate.The lower H+-ATPase activity was considered as a key factor which inhibited the root cell elongation under alkaline stress.However,theexpression level of plasma membrane H+-ATPase genes were not influenced by externalpH and we found overexpressing the OsA8 in rice significantly promoted the root elongation rate under a normal pH,but no effect on rice adaption to alkaline stress,indicating that the root elongation was inhibited by the low H+-ATPase activity,but the the activity was not regulated by the transcriptional level of OsAs genes.We also noted that ethylene was involved in alkaline stress-induced inhibition of rice growth.We explored the relationship between ethylene and H+-ATPase activity by monitoring root medium acidification and H+ efflux rate with exogenous Ag+ and ACC treatment.Ethylene negatively affected the root medium acidification and decreased the H+ efflux rate.Besides,the root elongation of ein2 mutants which showed less insensitive to alkaline stress,but was restricted by NaVO3.These results sufficiently indicated that H+-ATPase is involved in ethylene-mediated inhibition of rice growth and ethylene act as the upstream regulator negatively affected H+-ATPase activity.